RADseq, shotgun, and PacBio DNA extraction and genomic sequencing for B. microptera

We used population genomics within B. microptera from the eastern Pacific to explore the evolution and connectivity of a species (Table 2). We used the E.Z.N.A. Mollusk DNA kit (Omega Bio-Tek, Norcross, GA) following the manufacturer’s protocol to extract genomic DNA from 67 specimens for restriction site-associated DNA (RAD) or whole-genome shotgun sequencing. All individuals that were sequenced for SNP calling were also Sanger-sequenced at all targeted loci (SI Supplementary Appendix A2; Supplementary Table S1). DNA was quantified using the DNA High-Sensitivity assay with the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, United States). Sixteen of the individuals were used to prepare ezRADseq libraries based on modified methods (Toonen et al., 2013) fully described in (Breusing et al., 2019). As in Breusing et al., 2019, libraries were sequenced on a HiSeq 2,500 instrument at UC Davis, with a paired end 2 x 150 bp protocol. Shotgun sequencing libraries were prepared for the remainder of the 51 individuals, eighteen with a TruSeq Nano kit (Illumina Inc., San Diego, CA), and sequenced using the same protocol as the ezRAD libraries. The remainder were prepared with the Nextera XT DNA Library Preparation Kit (Illumina Inc., San Diego, CA) for lower input DNA, and sequenced on the HiSeq 4,000 instruments at UC Davis (Davis, CA) and MedGenome Labs (Foster City, CA). Four individuals were used in both ezRAD and shotgun sequencing to test for cross-platform discrepancies.

Programs, versions, tests and parameters estimated from WGS data.

An F3 cultured individual of B. microptera from the Monterey Bay Aquarium was photographed, illustrated, frozen whole in liquid nitrogen, then used to construct a PacBio CLR library, which was sequenced on a Sequel II on November 18, 2019 (D. Schultz, in prep, GenBank# {"type":"entrez-nucleotide","attrs":{"text":"JAIOUN000000000","term_id":"2328015198","term_text":"JAIOUN000000000"}}JAIOUN000000000). The F3 animals in the culture were collected in December 2018. Statistical programs further detailed in Table 2.